Project Summary/Abstract Inflammasomes are multi-protein complexes that assemble in the cytosol in response to infection or other noxious stimuli. Inflammasomes serve as platforms to activate inflammatory caspase proteases such as Caspase-1. Active Caspase-1 initiates inflammation by cleaving pro-IL1B and pro-IL18 cytokines into their active and secreted forms. Active Caspase-1 can also trigger a lytic form of cell death, called pyroptosis, by cleaving and activating a pore-forming protein called Gasdermin D. Inflammasomes play critical roles in initiating host defense against diverse viral, bacterial, fungal and protozoan pathogens. In addition, genetic evidence from humans and mice has established that inappropriate inflammasome activation can cause severe autoimmune and inflammation-driven diseases. However, the molecular mechanisms of how inflammasomes detect pathogens and become activated remain poorly understood. This proposal focuses on the NLRP1 inflammasome, which appears to have a novel but poorly understood mechanism of activation. Prior studies have demonstrated that mouse NLRP1 is activated by a toxin produced by Bacillus anthracis, but how this occurs and whether there are pathogen-encoded activators of human NLRP1 remains unclear. To address these gaps in our knowledge, this proposal has two aims: 1. Determine the biochemical mechanism of NLRP1 activation; 2. Identify novel pathogen-associated enzymes that activate NLRP1. Completion of these aims will increase our understanding of the mechanisms by which the innate immune system detects and responds to pathogens.